Skip to main content
SpringerLink
Log in

Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Latgé, J. P. (2007). The cell wall: carbohydrate armor for the fungal cell. Molecular Microbioogy, 66, 279–290.

    Article  Google Scholar 

  2. Cabib, E., & Arroyo, J. (2013). How carbohydrates sculpt cells: chemical control of morphogenesis in the yeast cell wall. Nature Reviews in Microbiology, 11, 648–655.

    Article  CAS  Google Scholar 

  3. Cosgrove, D. J. (2005). Growth of the plant cell wall. Nature Reviews in Molecular and Cell Biology, 6, 850–861.

    Article  CAS  Google Scholar 

  4. Fry, S. C. (2004). Primary cell wall metabolism: tracking the careers of wall polymers in living plant cells. New Phytologist, 161, 641–675.

    Article  CAS  Google Scholar 

  5. Ait-Mohand, F., & Farkaš, V. (2006). Screening for hetero-transglycosylating activities in extracts from nasturtium (Tropaeolum majus). Carbohydrate Research, 341, 577–581.

    Article  Google Scholar 

  6. Hrmova, M., Farkaš, V., Lahnstein, J., & Fincher, G. B. (2007). A barley xyloglucan xyloglucosyl transferase covalently links xyloglucan, cellulosic substrates, and (1, 3; 1, 4)-β-D-glucans. Journal of Biological Chemistry, 282, 12951–12962.

    Article  CAS  Google Scholar 

  7. Frankova, L., & Fry, S. C. (2013). Biochemistry and physiological roles of enzymes that ‘cut and paste’ plant cell-wall polysaccharides. Journal of Experimental Botany, 64, 3519–3550.

    Article  CAS  Google Scholar 

  8. Fry, S. C., Smith, R. C., Renwick, K. F., Martin, D. J., Hodge, S. K., & Matthews, K. J. (1992). Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. Biochemical Journal, 282, 821–828.

    Article  CAS  Google Scholar 

  9. Thompson, J. E., & Fry, S. C. (2001). Restructuring of wall‐bound xyloglucan by transglycosylation in living plant cells. The Plant Journal, 26, 23–34.

    Article  CAS  Google Scholar 

  10. Stratilová, E., Ait-Mohand, F., Řehulka, P., Garajová, S., Flodrová, D., Řehulková, H., & Farkaš, V. (2010). Xyloglucan endotransglycosylases (XETs) from germinating nasturtium (Tropaeolum majus) seeds: isolation and characterization of the major form. Plant Physiology and Biochemistry, 48, 207–215.

    Article  Google Scholar 

  11. Johnston, S. L., Prakash, R., Chen, N. J., Kumagai, M. H., Turano, H. M., Cooney, J. M., & Schröder, R. (2013). An enzyme activity capable of endotransglycosylation of heteroxylan polysaccharides is present in plant primary cell walls. Planta, 237, 173–187.

    Article  CAS  Google Scholar 

  12. Schröder, R., Atkinson, R. G., & Redgwell, R. J. (2009). Re-interpreting the role of endo-β-mannanases as mannan endotransglycosylase/hydrolases in the plant cell wall. Annals of Botany, 104, 197–204.

    Article  Google Scholar 

  13. Fry, S. C., Mohler, K. E., Nesselrode, B. H., & Frankova, L. (2008). Mixed‐linkage β‐glucan: xyloglucan endotransglucosylase, a novel wall‐remodelling enzyme from Equisetum (horsetails) and charophytic algae. The Plant Journal, 55, 240–252.

    Article  CAS  Google Scholar 

  14. Gastebois, A., Mouyna, I., Simenel, C., Clavaud, C., Coddeville, B., Delepierre, M., Latgé, J. P., & Fontaine, T. (2010). Characterization of a new β(1-3)-glucan branching activity of Aspergillus fumigatus. Journal of Biological Chemistry, 285, 2386–2396.

    Article  CAS  Google Scholar 

  15. Hartland, R. P., Fontaine, T., Debeaupuis, J. P., Simenel, C., Delepierre, M., & Latgé, J. P. (1996). A Novel β-(1, 2, 3)-Glucanosyltransferase from the Cell Wall of Aspergillus fumigatus. Journal of Biological Chemistry, 271, 26843–26849.

  16. Ragni, E., Fontaine, T., Gissi, C., Latge, J. P., & Popolo, L. (2007). The Gas family of proteins of Saccharomyces cerevisiae: characterization and evolutionary analysis. Yeast, 24, 297–308.

    Article  CAS  Google Scholar 

  17. Kovacova, K., Degani, G., Stratilova, E., Farkas, V., & Popolo, L. (2015). Catalytic properties of Phr family members of cell wall glucan remodeling enzymes: implications for the adaptation of Candida albicans to ambient pH. FEMS Yeast Research, 15, 1–15.

    Article  Google Scholar 

  18. Cabib, E., Farkas, V., Kosik, O., Blanco, N., Arroyo, J., & McPhie, P. (2008). Assembly of the yeast cell wall Crh1p and Crh2p act as transglycosylases in vivo and in vitro. Journal of Biological Chemistry, 283, 29859–29872.

    Article  CAS  Google Scholar 

  19. Mazáň, M., Blanco, N., Kováčová, K., Firáková, Z., Řehulka, P., Farkaš, V., & Arroyo, J. (2013). A novel fluorescence assay and catalytic properties of Crh1 and Crh2 yeast cell wall transglycosylases. Biochemical Journal, 455, 307–318.

    Article  Google Scholar 

  20. Plaine, A., Walker, L., Da Costa, G., Mora-Montes, H. M., McKinnon, A., Gow, N. A., & Richard, M. L. (2008). Functional analysis of Candida albicans GPI-anchored proteins: roles in cell wall integrity and caspofungin sensitivity. Fungal Genetics and Biology, 45, 1404–1414.

    Article  CAS  Google Scholar 

  21. Calderon, J., Zavrel, M., Ragni, E., Fonzi, W. A., Rupp, S., & Popolo, L. (2010). PHR1, a pH-regulated gene of Candida albicans encoding a glucan-remodelling enzyme, is required for adhesion and invasion. Microbiology, 156, 2484–249.

    Article  CAS  Google Scholar 

  22. Trujillo, R. (1968). Preparation of carboxymethylchitin. Carbohydrate Research, 74, 483–485.

    Article  Google Scholar 

  23. Sulova, Z., Lednicka, M., & Farkas, V. (1995). A colorimetric assay for xyloglucan-endotransglycosylase from germinating seeds. Analytical Biochemistry, 229, 80–85.

    Article  CAS  Google Scholar 

  24. Kosik, O., & Farkas, V. (2008). One-pot fluorescent labeling of xyloglucan oligosaccharides with sulforhodamine. Analytical Biochemistry, 375, 232–236.

    Article  CAS  Google Scholar 

  25. Mazáň, M., Ragni, E., Popolo, L., & Farkaš, V. (2011). Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell wall biogenesis as determined by a novel fluorescent assay. Biochemical Journal, 438, 275–282.

    Article  Google Scholar 

  26. Gray, C. J., Weissenborn, M. J., Eyers, C. E., & Flitsch, S. L. (2013). Enzymatic reactions on immobilised substrates. Chemical Society Reviews, 42, 6378–6405.

    Article  CAS  Google Scholar 

  27. Frankova, L., & Fry, S. C. (2015). A general method for assaying homo‐and hetero‐transglycanase activities that act on plant cell‐wall polysaccharides. Journal of Integrative Plant Biology, 57, 411–428.

    Article  CAS  Google Scholar 

  28. Kosik, O., Auburn, R. P., Russell, S., Stratilova, E., Garajová, S., Hrmova, M., & Farkas, V. (2010). Polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts. Glycoconjugate Journal, 27, 79–87.

    Article  CAS  Google Scholar 

  29. Wang, D., Liu, S., Trummer, B. J., Deng, C., & Wang, A. (2002). Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Nature Biotechnology, 20, 275–281.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are thankful to Prof. Javier Arroyo (Universidad Complutense, Madrid, Spain) for kindly providing the Crh1p enzyme as well as Pichia stipitis strains transformed with Saccharomyces cerevisiae CRH1 and CRH2 genes and to Prof. Laura Popolo (University of Milano, Italy) for providing Pichia strains transformed with Candida albicans PHR1 and PHR2 genes and with Saccharomyces cerevisiae GAS1. The work was supported by grant no. 2/0020/12 from the Grant Agency for Science VEGA (Slovakia).

Author information

Authors and Affiliations

  1. Institute of Chemistry, Centre for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, 84538, Bratislava, Slovakia

    Kristína Kováčová & Vladimír Farkaš

Authors
  1. Kristína Kováčová
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vladimír Farkaš
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vladimír Farkaš.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

ᅟ (DOCX 112 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kováčová, K., Farkaš, V. Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases. Appl Biochem Biotechnol 178, 1652–1665 (2016). https://doi.org/10.1007/s12010-015-1973-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1973-8

Keywords

Search

Navigation